We recently performed studies to reduce the size of Inf2 to determine whether additional candidate genes emerged, and identified Tnf, Lta, Hsp70, and MHC class II genes as important determinants of susceptibility to ozone (O3)-induced airway inflammation. Ongoing studies are investigating the roles of Notch3 and Notch4, also located in Inf2, in O3-induced inflammation. Notch receptors are cell surface receptors important in development and immune cell differentiation. To test the roles of these genes in the model, wild type (B6129SF1, WT), Notch3 (Notch3-/-) and Notch4 (Notch4-/-) knockout mice were exposed to 0.3 ppm O3 or filtered air continuously for 6-48 hr. Immediately after exposure, airway inflammation and injury was assessed using protein concentration (a marker of lung permeability) and inflammatory cells in bronchoalveolar lavage fluid (BALF). O3 significantly increased BALF protein in all genotypes, but greater concentrations were found in Notch3-/- compared to WT (24, 48 hr), and concentrations were greater in Notch4-/- mice compared to Notch3-/- (24, 48 hr). Greater mean numbers of BALF neutrophils were found in Notch3-/- and Notch4-/- mice compared to WT (24, 48 hr). Expression of whole lung Tnf was significantly increased after O3 (24 hr) in all genotypes, and was greater in Notch3-/- and Notch4-/- compared to WT. Pre-treatment with the TNF inhibitor etanercept significantly attenuated the enhanced O3-induced BALF neutrophils in Notch3-/- and Notch4-/- relative to WT. O3-induced transcript expression of other Inf2 genes was not different between genotypes. We then used mRNA transcriptomics analyses to further investigate the role of Notch3/4. Statistical and visual data mining approaches identified differentially expressed genes basally e.g. Gbp1, Cntn1 and after O3 e.g. Ccl7, Il33 between WT and KO mice. Results are consistent with the hypothesis that Notch3 and Notch4 are susceptibility genes for O3-induced airway inflammation. Furthermore, results suggest an important interaction between Notch3, Notch4, and Tnf. These novel findings suggest Notch receptors protect against the innate immune inflammatory response to O3. In another study we have tested the hypothesis that the gene mannose binding lectin (MBL), which has a central role in the activation of the complement pathway of innate immunity, is necessary to elicit some of the pro-inflammatory events caused by ozone-mediated activation of the innate immune system. Our in vivo studies have shown, compared to wild type mice (Mbl+/+), there was significantly less neutrophilic infiltration in the lungs of mice with targeted deletion of Mbl (Mbl-/-) exposed to O3 (0.3 ppm) for 72 hours. We also found reduced levels of the neutrophil attractants MIP-2 and LIX at 48 hours post-exposure in Mbl-/- mice compared to Mbl+/+ mice. Microarray analyses have identified basal and post-exposure profiles and expression response profiles that differ between Mbl+/+ and Mbl-/- mice, providing insight to the mechanisms through which MBL modulates the pulmonary response to O3 inhalation. These novel studies are the first to identify a role for MBL in response of the lung to oxidative stress, and should lead to a better understanding of mechanisms of susceptibility. Children may be more at risk to air pollution than adults due to higher minute ventilation rates and activity levels outdoors, and continued lung development into adolescence. To study the impact of O3 exposure on the developing lung, we have collaborated with Dr. Edward Postlethwait (Univ Alabama, Birmingham) to study O3 effects on gene expression in infant rhesus macaque monkeys that were exposed to a regimen mimicking urban conditions and site and exposure duration samples were obtained for gene expression analysis. Primates were raised in filtered air (FA) and nighttime exposures to 0.5 ppm O3 conducted for 1 cycle (9 d FA followed by 8 hrs/d O3 for 5 d), 11 cycles, or FA. Exposures ended at 180 d of age. Immediately post exposure, lungs were microdissected to obtain central axial airways (generation 8-10) and terminal bronchioles devoid of parenchyma, and stored in RNAlater. RNA was pooled to provide a single sample for each experimental group. Gene expression (Agilent rhesus monkey oligo microarrays) was analyzed initially by K-means clustering. Informative patterns were analyzed (Ingenuity) to identify interaction between differentially expressed genes. A number of informative patterns were identified. 1) Genes upregulated in axial and terminal bronchiole tissue after 1 cycle: inflammatory and immune responses (e.g. IL8, TNF). 2) Upregulated genes in axial tissue after 11 cycles: cellular inflammation and hematological system (IL1B, IL17, MAPK). 3) Genes differentially expressed in axial and terminal bronchioles irrespective of O3 exposure: developmental (ACAN, MUC2, CX3CL1) and immune function (IL17RD, FGF1, DAP1). In infant primates, the superimposition of injury and repair on growth and development results in anatomic and exposure specific alterations in gene expression that is likely coupled to the O3-induced structural, inflammatory, and biochemical effects also observed. Asthma is a known risk factor for acute ozone-associated respiratory disease. Ozone causes an immediate decrease in lung function and increased airway inflammation. The role of atopy and asthma in modulation of ozone-induced inflammation has not been determined. We sought to determine whether atopic status modulates ozone response phenotypes in human subjects. Fifty volunteers (25 healthy volunteers, 14 atopic nonasthmatic subjects, and 11 atopic asthmatic subjects not requiring maintenance therapy) underwent a 0.4-ppm ozone exposure protocol. Ozone response was determined based on changes in lung function and induced sputum composition, including airway inflammatory cell concentration, cell-surface markers, and cytokine and hyaluronic acid concentrations. All cohorts experienced similar decreases in lung function after ozone. Atopic and atopic asthmatic subjects had increased sputum neutrophil numbers and IL-8 levels after ozone exposure; values did not significantly change in healthy volunteers. After ozone exposure, atopic asthmatic subjects had significantly increased sputum IL-6 and IL-1beta levels and airway macrophage Toll-like receptor 4, FcepsilonRI, and CD23 expression; values in healthy volunteers and atopic nonasthmatic subjects showed no significant change. Atopic asthmatic subjects had significantly decreased IL-10 levels at baseline compared with healthy volunteers; IL-10 levels did not significantly change in any group with ozone. All groups had similar levels of hyaluronic acid at baseline, with increased levels after ozone exposure in atopic and atopic asthmatic subjects. Atopic asthmatic subjects have increased airway inflammatory responses to ozone. Increased Toll-like receptor 4 expression suggests a potential pathway through which ozone generates the inflammatory response in allergic asthmatic subjects but not in atopic subjects without asthma